Endocytosis is a principal means by which cells regulate plasma membrane composition, which in turn is critical for determining cellular physiology. Two basic endocytic pathways have been described, one involves the protein clathrin while the other appears to function in the absence of clathrin. Though many of the components that participate in clathrin dependent endocytosis have been identified, their function and regulation are still not well understood. Even less is known about the components involved in clathrin-independent endocytosis. The long term goal of the studies described here is to understand the process of endocytosis at the molecular level. A novel Fluorescence-Activated Cell Sorter (FACS)-based screen was used to uncover the essential yeast gene PAN1. PAN1 is required for endocytosis and exhibits genetic and/or physical interactions with several other genes that encode proteins known to be required for endocytosis. The primary amino acid sequence of Pan1p suggests that it has a three-domain structure in which each domain participates in distinct protein-protein interactions. Pan1p may serve a central role in clathrin-dependent endocytic pathway by integrating the functions of distinct cellular events (proteosome-independent ubiquitination, actin polymerization, and clathrin-mediated processes) that are all required for efficient endocytosis. To test this hypothesis, interactions between Pan1p and other known endocytic proteins will be analyzed using genetic, biochemical, morphological and functional assays. In addition, the function and regulation of a novel Pan1p interaction partner, Ent1p, will also be examined. Finally, the FACS-based screen will be used to uncover new factors required not only for clathrin-dependent endocytosis, but also for the clathrin-independent pathway. These studies should define the functional role of Pan1p in the endocytic process, as well as identify new yeast proteins with human homologues that will serve as targets for therapeutics in human disease such as leukemia or breast cancer.